Agents and methods for treating cbreb binding proteins-dependent cancers

ABSTRACT

Single chain peptides comprising either a cell penetrating HIV-TAT peptide sequence and a MYB:CBP complex interfering peptide sequence from MYB, or comprising a cell penetrating HIV-TAT peptide sequence, a CBP binding peptide sequence from CREB and a MYB:CBP complex interfering peptide sequence from MYB, are provided for use in preventing MYB:CBP complex formation and downstream events leading to cancer, in particular a leukemia. Both L-amino acid single chain peptides and retro-inverso single chain peptides are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is continuation of U.S. patent application Ser. No.16/346,834, filed May 1, 2019 and issued as U.S. Pat. No. 11,208,446,which is a U.S. National Phase Application of PCT InternationalApplication No. PCT/US2017/059579, international filing date Nov. 1,2017, claiming the benefit of U.S. Provisional Application No.62/415,800, filed Nov. 1, 2016, which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

In spite of substantial efforts, therapy of pediatric and adult acutemyeloid leukemia (AML) has not had significant improvements over thelast 30 years with outcomes particularly poor for patients withhigh-risk molecular features and those whose disease is resistant tointensive combination chemotherapy. Both myeloid and lymphoblasticleukemias depend on the aberrant activity of key molecular regulators ofgene transcription. Central to this molecular mechanism is thetranscription factor MYB, which is a sequence-specific DNA bindingprotein that trans-activates expression of genes important for leukemiacell growth and survival. Among MYB's various binding partners, CBP, orCREB-binding protein, is an important co-activator that interacts withthe transactivation domain of MYB and exerts histone acetyltransferaseactivity fundamental in downstream gene regulation.

MYB has been implicated as a human leukemia oncogene, among othercancers, with abnormalities in MYB seen in various leukemias, both acuteand chronic. In addition to point mutations and truncation mutations inMYB, there is evidence of overexpression of MYB in ALL. MYB has beenrepeatedly identified as a molecular requirement for the initiation andmaintenance of a wide variety of AML subtypes in xenografts andgenetically-engineered mouse leukemia models. MYB particularly plays animportant role in the leukemogenic maintenance of MLL-rearrangedleukemias, as shown by the marked survival in vivo of mice with aninducible MYB shRNA knock down system. In an engineered mouse model thatassociates a requirement of the MYB:CBP complex in the initiation ofAML, Myb mutant cells harboring a E308G mutation in the MYB:CBPinterface were unable to transform to AML with either the AML1-ETO orMLL-AF9 oncogenes.

Translation of this knowledge into therapies to block leukemogenic MYBactivity is hindered by the pharmacologic challenges of targetingtranscription factors and protein-protein interactions. The necessaryintranuclear physical interactions between transcription factors andtheir co-activators provides a site-specific mark for potential drugtargets.

It is towards new means of blocking leukemogenic MYB activity as well asfor the treatment of various cancers that the present invention isdirected.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, the invention is directed to a single chain peptidecomprising a cell penetrating peptide sequence from HIV-TAT and a CBPbinding peptide sequence from MYB. In one embodiment the single chainpeptide is comprised of L-amino acids. In one embodiment the sequencefrom HIV-TAT is at the N-terminal part of the single chain peptide, andthe sequence from MYB is at the C-terminal part. In one embodiment, thesequence in the single chain peptide from HIV-TAT is L-amino acidsequence YGRKKRRQRRR (SEQ ID NO:16). In one embodiment, the sequence inthe single chain peptide from MYB is L-amino acid sequenceKRIKELELLLMSTENELK (SEQ ID NO:18). In one embodiment, the cellpenetrating peptide sequence from HIV-TAT and the CBP binding peptidesequence from MYB are joined by a linker. In one embodiment the linkercomprises one or more amino acids. In one embodiment, the linker is GG.

In one embodiment, the single chain peptide has the L-amino acidsequence YGRKKRRQRRRGGKRIKELELLLMSTENELK (SEQ ID NO:2).

In one embodiment, the cell penetrating peptide sequence from HIV-TATand the CBP binding peptide sequence from MYB are comprised of D-aminoacids, and the order of amino acids is inverted from that in the L-aminoacid sequence (i.e., a retro-inverso sequence). In one embodiment, theretro-inverso sequence from MYB is at the N-terminal part of the singlechain peptide, and the retro-inverso sequence from HIV-TAT is at theC-terminal part. In one embodiment, the cell penetrating peptidesequence from HIV-TAT is the D-amino acid sequence RRRQRRKKRGY (SEQ IDNO:17). In one embodiment, the CBP binding peptide sequence from MYB isthe D-amino acid sequence KLENETSMLLLELEKIRK (SEQ ID NO:19). In oneembodiment, the cell penetrating peptide sequence from HIV-TAT and theCBP binding peptide sequence from MYB are joined by a linker. In oneembodiment the linker comprises one or more amino acids. In oneembodiment, the linker is GG.

In one embodiment, the single chain peptide has the D-amino acidsequence KLENETSMLLLELEKIRKGGRRRQRRKKRGY (SEQ ID NO:1).

In one embodiment, each of the cell penetrating peptide sequence fromHIV-TAT, the CBP binding peptide sequence from MYB peptide, or both, inthe single chain peptide comprises one or more amino acid additions,deletions or substitutions, or the N-terminus is acetylated, theC-terminus is amidated, or any combination thereof. In one embodiment,each of the cell penetrating peptide sequence from HIV-TAT, the CBPbinding peptide sequence from MYB, or both, in the single chain peptideindependently has at least 98%, at least 95%, at least 90%, at least85%, at least 80% or at least 75% sequence identity with the respectiveHIV-TAT and MYB sequences within SEQ ID NOS: 1 and 2.

In one embodiment, the single chain peptide has the L-amino acidsequence [Ac]-YGRKKRRQRRRGGKRIKELELLLMSTENELK-[NH2] (SEQ ID NO:2) and inone embodiment, the single chain peptide has the D-amino acid sequence[Ac]-KLENETSMLLLELEKIRKGGRRRQRRKKRGY-[NH2] (SEQ ID NO:1). [Ac]represents an acetyl group at the N terminus, and [NH2] represents anamide at the C terminus.

In one embodiment, one of the sequence from HIV-TAT or the sequence fromMYB comprises L-amino acids, and the other sequence comprises D-aminoacids in the reverse order (i.e., retro-inverso).

In one embodiment, the invention is directed to a single chain peptidecomprising a cell penetrating peptide sequence from HIV-TAT, a CBPbinding peptide sequence from CREB, and a CBP binding peptide sequencefrom MYB. In one embodiment the single chain peptide is comprised ofL-amino acids. In one embodiment the sequence from HIV-TAT is at theN-terminal part of the single chain peptide, the sequence from MYB is atthe C-terminal part, and the sequence from CREB is between them. In oneembodiment, the sequence in the single chain peptide from HIV-TAT is theL-amino acid sequence YGRKKRRQRRR (SEQ ID NO:16). In one embodiment, thesequence of the CBP binding peptide sequence from CREB is the L-aminoacid sequence RREILSRRPpSYRK (SEQ ID NO:20), and the sequence in thesingle chain peptide from MYB is L-amino acid sequence LELLLMSTENELK(SEQ ID NO:21). In one embodiment, the cell penetrating peptide sequencefrom HIV-TAT and the CBP binding peptide sequence from MYB are joined bya linker. In one embodiment, the linker is GG. The CBP binding peptidesequence from MYB and the CBP binding peptide sequence from CREB mayalso be joined by a linker. In one embodiment, the order of thesesequences in the L-amino acid, single-chain peptide is, from the N-to-Cterminus, is the sequence from HIV-TAT, the sequence from CREB, sequencefrom MYB. In another embodiment, the order is the L-amino acids is thesequence from HIV-TAT, the sequence from MYB and the sequence from CREB.

In one embodiment, the single chain peptide has the L-amino acidsequence YGRKKRRQRRRGGRREILSRRPpSYRKLELLLMSTENELK (SEQ ID NO:22).

In one embodiment, the cell penetrating peptide sequence from HIV-TAT,the CBP binding peptide sequence from CREB, and the CBP binding peptidesequence from MYB are composed of D-amino acids, and the order of aminoacids is reversed from that of the L-amino acid sequence (i.e., aretro-inverso peptide). In one embodiment, the sequence from HIV-TAT isthe D-amino acid sequence RRRQRRKKRGY (SEQ ID NO:17). In one embodimentthe CBP binding peptide sequence from CREB is the D-amino acid sequenceKRYpSPRRSLIERR (SEQ ID NO:23). In one embodiment, the CBP bindingpeptide sequence from MYB is the D-amino acid sequence KLENETSMLLLEL(SEQ ID NO:24). In one embodiment, the order of these sequences in thesingle-chain peptide is, from the N-to-C terminus, is the retro-inversosequence from MYB, the retro-inverso sequence from CREB, and theretro-inverso sequence from HIV-TAT. In another embodiment, the orderfrom N-to-C is the retro-inverso sequences is the sequence from CREB,the sequence from MYB, and the sequence from HIV-TAT.

In one embodiment, the single chain peptide has the D-amino acidsequence KLENETSMLLLELKRYpSPRRSLIERRGGRRRQRRKKRGY (SEQ ID NO:25).

In one embodiment, one of the sequence from HIV-TAT, the sequence fromMYB, and the sequence from CREB is comprised of L-amino acids, and theother sequences comprise retro-inverso sequences. In another embodiment,two among the sequence from HIV-TAT, the sequence from MYB, and thesequence from CREB are comprised of L-amino acids, and the othersequence is retro-inverso.

In one embodiment, each of the cell penetrating peptide sequence fromHIV-TAT, the CBP binding peptide sequence from CREB, and the CBP bindingpeptide sequence from MYB peptide, or two or three of these sequenceswithin the single chain peptide, comprises one or more amino acidadditions, deletions or substitutions, or the N-terminus is acetylated,the C-terminus is amidated, or any combination thereof. In oneembodiment, each of the cell penetrating peptide sequence from HIV-TAT,the CBP binding peptide sequence from CREB, the CBP binding peptidesequence from MYB, or two or three of these sequences within the singlechain peptide, independently has at least 98%, at least 95%, at least90%, at least 85%, at least 80% or at least 75% sequence identity withthe respective sequence of the HIV-TAT, CREB, and MYB sequences withinSEQ ID NOS: 16, 20 and 18, respectively. Furthermore, the single chainpeptide of this embodiment may comprise longer or shorter fragments ofthe three respective components, as further described below. Theembodiments apply to either the L-amino acid or D-amino acid/reversedsequences of the invention.

In one embodiment, the single chain peptide has the L-amino acidsequence [Ac]-YGRKKRRQRRRGGRREILSRRPpSYRKLELLLMSTENELK-[NH2] (SEQ IDNO:26) and in one embodiment, the single chain peptide has the D-aminoacid sequence [Ac]-KLENETSMLLLELKRYpSPRRSLIERRGGRRRQRRKKRGY-[NH2] (SEQID NO:27). In different embodiments, the N-terminus may be acetylated,the C-terminus amidated, or the combination of both. [Ac] represents anacetyl group at the N terminus, and [NH2] represents an amide at the Cterminus.

In another embodiment nucleic acid molecules encoding any of thepeptides of the invention are provided, as well as vectors comprisingthe nucleic acid sequence of a peptide of the invention.

In another embodiment a composition is provided comprising the singlechain peptide of any of the foregoing embodiments, and a carrier,excipient or diluent.

In another embodiment, a method for treating cancer is providedcomprising administering to a patient in need thereof an effectiveamount of the foregoing composition or a single chain peptide of any ofthe foregoing embodiments. In one embodiment, the cancer is acutelymphoblastic leukemia or acute lymphocytic leukemia, acute myeloidleukemia, or chronic myeloid leukemia (CML). In other embodiments, thecancer is lymphoma, small cell lung cancer, renal cell carcinoma,adenoid cystic carcinoma, squamous cell carcinoma of the head and neck,neuroblastoma, pancreatic cancer, follicular lymphoma, mantel celllymphoma, breast cancer, uterine cancer, ovarian cancer, hepatocellularcarcinoma, lung cancer, germ cell tumor, non-small cell lung cancer,gastric cancer, renal cancer, Kaposi's sarcoma, mesothelioma,desmoplastic small round cell tumor, Ewing sarcoma or lungadenocarcinoma.

These and other aspects of the invention will be understood from thefollowing brief description of the figures and detailed description ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a-d show that a compound of the invention competes with theMYB:CBP complex in acute myeloid leukemia. FIG. 1a is a MolecularDynamic Simulation modeling was performed to compare the molecularinteractions within the native MYB:CBP complex to the interactionbetween a MYB peptide to CBP (L-amino acid peptide, labeled as L-aa (SEQID NO:2), and D-amino acid, labeled MYBMIM, SEQ ID NO:1, are comparedhere). FIG. 1b is a Microscale Thermopheresis (MST) to analyze thebinding affinity of MYB peptides to the purified CBP-KIX domain. FIG. 1cshows the intranuclear penetration of FITC-labelled TAT peptides inMOLM13 cells using confocal microscopy. FIG. 1d is a Western blotshowing specific binding of biotinylated-MYBMIM to CBP in MV411 humanAML cells.

FIG. 2a-g show that a compound of the invention downregulatesMYB-regulated genes and induces apoptosis. FIG. 2a is the quantificationof live AML cells using Trypan blue exclusion after MYBMIM treatment.FIG. 2b shows induction of apoptosis observed with MYBMIM treatment ofhuman AML cells. Flow cytometric analysis of Annexin-V and DAPI stainingof MOLM13 cells after treatment with TG3 (20 μM) and MYBMIM (20 μM) for24 hours. FIG. 2c shows that MYBMIM induces cell death withoutmorphologic evidence of differentiation. FIG. 2d is a heatmap showingMYBMIM-induced gene expression changes in MOLM13 cells after 6 hourtreatment with TG3 (20 μM) and MYBMIM (20 μM). FIG. 2e is a volcano plotof MYBMIM treated MV411 human AML cells compared to control. FIG. 2fshows BCL2 and MYC mRNA expression was measured by RT-qPCR andnormalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)expression. FIG. 2g shows ectopic expression of MSCV-Ires-GFP vectorcontaining BCL2 in MV411 cells partially rescues MYBMIM-inducedapoptosis, shown here by measurement of ATP activity using a CellTiterGlo luminescence assay.

FIGS. 3a-d show that a compound of the invention exhibits anti-leukemiaefficacy in vivo FIG. 3a shows that MYBMIM has no significant effect oncolony formation of CD34-enriched hematopoietic progenitor cellsisolated from human umbilical cord blood and grown in growth-factorenriched semi-solid media. FIG. 3b shows white blood cell counts,measured in thousands/μL and FIG. 3c shows hemoglobin, measured in g/dL,n=5 for each treatment group. FIG. 3d shows the survival analysis ofprimary patient-derived MLL-rearranged leukemia cells (5×10{circumflexover ( )}5 cells per mouse via tail vein injection) engrafted intosublethally irradiated immunodeficient mice and treated with MYBMIM viaintraperitoneal injection for 14 days (indicated as Treatment from days3-17), p=0.0038.

FIGS. 4a-d show the activity of peptides of the invention against apanel of AML cell lines, and cell viability against MV411 cells. FIG. 4ashows SEQ ID NO:1 (MYBMIM) and FIG. 4b shows SEQ ID NO:25 (CRYBMIM)dosed at 10 and 20 μM every 48 hours for a 6 day period. FIG. 4c showsSEQ ID NO:1 and FIG. 4d shows SEQ ID NO:25 tested at 20 μM dose across apanel of AML cell lines and the results from day 6 of treatment

DETAILED DESCRIPTION OF THE INVENTION

Despite recent efforts to improve stratification of conventionalchemotherapy for the treatment of patients with acute myeloid leukemia(AML), cure rates remain poor. Recent genomic profiling studies havebegun to reveal that AML is characterized by the predominance ofmutations of genes encoding regulators of gene transcription andchromatin structure. Indeed, most AML chromosomal translocations, suchas those involving MLL gene rearrangements, encode chimerictranscription or chromatin remodeling factors. Recent functional genomicefforts have identified specific molecular dependencies of aberrant AMLgene expression, such as for example the requirement of DOT1L for themaintenance of MLL-rearranged leukemias, prompting the clinicaldevelopment of DOT1L methyltransferase inhibitors for AML therapy.Similarly, additional AML subtypes appear dependent on aberrant geneexpression, conferring a susceptibility to inhibition of CDK8 and BRD4that regulate the Mediator transcriptional coactivator complex.

In addition to these established mechanisms, recent studies have alsoimplicated aberrant activity of hematopoietic transcription factors andtheir coactivators, such as MYB and CREB-binding protein (CBP), in AMLpathogenesis. In particular, MYB is a sequence-specific hematopoietictranscription factor that is required for the survival of AML1-ETO andMLL-rearranged leukemias. Transient suppression of MYB expressioneliminates MLL-AF9 leukemias but is dispensable for normal myelopoiesis,emphasizing its specific functional requirements in AML pathogenesis.Leukemogenic activities of MYB requires its physical and specificassociation with the transcriptional co-activator CBP. This interactionis associated with recruitment of CBP and its chromatin remodeling oftranscriptional circuits required for leukemogenesis. Indeed, Booreanamice mutant for Myb E308G that affects the molecular recognition of theKIX domain of CBP by MYB exhibit normal hematopoiesis, but are resistantto MLL-AF9-induced leukemogenesis.

Whereas previous attempts to interfere with aberrant transcriptionalcoactivation in AML have focused on pharmacologic blockade of CBPacetyltransferase activities, an alternative strategy is embodied hereinto dismantle the assembly of the leukemogenic transcriptionfactor-coactivator complex. The specific requirement of MYB E308 formolecular recognition of the CBP KIX domain was pursued. Using molecularmechanics simulations and structural analysis of the MYB:CBP molecularcomplex, stabilized, cell-penetrant peptidomimetic inhibitors of MYB:CBPbinding were created. Consequently, their molecular and cellularactivities, mechanisms of transcriptional regulation, and therapeuticactivities in preclinical leukemia models in vitro and in vivo wereinvestigated. These studies also support use of the same or similarstrategy in treatment of any cancers in which MYB:CBP interactionleading to cancer is undesirable.

In one embodiment, peptides of the invention comprise a single chainamino acid sequence comprising two sequences: one sequence derived fromthe HIV-TAT protein, which allows for cell membrane penetration, and theother sequence is an amino acid sequence derived from MYB, said MYBsequence capable of interacting with and interfering with the MYB:CBPinterface to prevent downstream activity of MYB:CBP such as that leadingto, for example, leukemogenesis, described elsewhere herein. The TATamino acid sequence and the MYB amino sequence on the single chainpeptide may be separated by an amino acid linker sequence. The TATsequence may be located at the N-terminal part of the single chain aminoacid sequence and the MYB sequence at the C-terminal part, or they maybe in the opposite positions, i.e., TAT at the C-terminal part and MYBat the N-terminal part. The single chain peptide may be comprised ofL-amino acids, the conventional forms of amino acids comprising mostnaturally-occurring peptides and proteins. Alternatively, the singlechain peptide may comprise D-amino acids, and the order of amino acidsin the sequence reversed from that of a peptide containing L-aminoacids, forming a “retro-inverso” form of the peptide, that retains theconformation and biological activities of the L-amino acid sequence, butis resistant to proteolysis in vivo. The ends of the single chainpeptide may be modified, such as but not limited to acetylation of theamino terminus and an amide present at the carboxy terminus. The startand stop amino acid in a description herein refers to the amino acidspositions in the L-amino acid sequence of the protein.

In one embodiment, the single-chain peptide comprises the TAT sequenceat the N-terminal part (the orientation when the peptide comprisesL-amino acids) and the MYB sequence at the C-terminal part. In thecorresponding retro-inverso sequence, the D-amino acid reversed orderTAT portion is at the C-terminus, and the D-amino acid, reversed orderMYB portion at the N-terminus. Such orientation of the TAT and MYBportions, in one embodiment, provide an increased biological activity ofthe single-chain peptide by better interfering with assembly of theMYB-CBP complex, reducing transactivation and thus negatively affectingdownstream gene regulation that would otherwise lead to dysregulation ofgene transcription and triggering leukemic cell growth. Furthermore, theTAT and MYB sequences within the single chain peptide may eachindependently be in the L-amino acid or D-amino acid/reversed (i.e.,retro-inverso) orientations, with or without a linker between thesequences. In one embodiment the linker comprises one or more aminoacids, such as GG.

In one embodiment, peptides of the invention comprise a single chainamino acid sequence comprising three sequences: one sequence derivedfrom the HIV-TAT protein, which allows for cell membrane penetration,one sequence from the CREB protein, capable of interacting with CBP, andone sequence is an amino acid sequence derived from MYB, said MYBsequence capable of interacting with and interfering with the MYB:CBPinterface to prevent downstream activity of MYB:CBP such as that leadingto, for example, leukemogenic, described elsewhere herein. The cellpenetrating peptide sequence from TAT and the CBP binding peptidesequence from CREB may be separated by an amino acid linker sequence.The CBP binding peptide sequence from CREB and the CBP binding peptidesequence from MYB on the single chain peptide may be separated by alinker. In one embodiment, the linker is GG. The cell penetratingpeptide sequence from TAT may be located at the N-terminal part of thesingle chain amino acid sequence and the MYB sequence at the C-terminalpart, or they may be in the opposite positions, i.e., the cellpenetrating peptide sequence from TAT at the C-terminal part and the CBPbinding peptide sequence MYB at the N-terminal part. In one embodimentthe linker comprises one or more amino acids, such as GG.

The single chain peptide may be comprised of L-amino acids, theconventional forms of amino acids comprising most naturally-occurringpeptides and proteins, or the single chain peptide may comprise D-aminoacids, and the order of amino acids in the sequence reversed, forming a“retro-inverso” form of the peptide, that retains the conformation andbiological activities of the L-amino acid sequence, but is resistant toproteolysis in vivo. The ends of the single chain peptide may bemodified, such as but not limited to acetylation of the amino terminusand an amide at the carboxy terminus. As will be noted below, each ofthe sequences from HIV-TAT, CREB, and MYB may be present in the singlechain peptide independently as an L-amino acid sequence or a D-aminoacid sequence in the reverse order of amino acids compared to theL-amino acid sequence.

In one embodiment, as noted above, the single-chain peptide of L-aminoacids comprises the cell penetrating peptide sequence from TAT at theN-terminal part and the MYB sequence at the C-terminal part, and theCREB sequence in between. In the corresponding retro-inverso sequence,the retro-inverso cell penetrating peptide from TAT sequence is at theC-terminus, and the retro-inverso CBP binding peptide sequence of MYB isat the N-terminus, and the retro-inverso CBP binding peptide sequencefrom CREB is between them. Such orientation of the “TAT”, “CREB” and“MYB” portions, in one embodiment, provide an increased biologicalactivity of the single-chain peptide by better interfering with assemblyof the MYB-CBP complex, reducing transactivation and thus negativelyaffecting downstream gene regulation that would otherwise lead todysregulation of gene transcription and triggering leukemic cell growth.Furthermore, the “TAT”, “CREB” and “MYB” sequences within the singlechain peptide may, as noted herein, each independently be in the L-aminoacid or D-amino acid/reversed (i.e., retro-inverso) orientations, withor without a linker between each sequence.

In another embodiment of the invention, modifications of one or moreamino acids in a single-chain peptide may be made, to retain or evenenhance the biological activity. In one embodiment, one or more aminoacids may be conservatively substituted. In another embodiment one ormore amino acids may be non-conservatively substituted. In suchsubstitutions, amino acids substitutions are possible provided thatthese do not excessively affect the biological activity of the peptide.In one embodiment the substitutions enhance biological activity.

As is well-known in the art, a “conservative substitution” of an aminoacid or a “conservative substitution variant” of a peptide refers to anamino acid substitution which maintains: 1) the secondary structure ofthe peptide; 2) the charge or hydrophobicity of the amino acid; and 3)the bulkiness of the side chain or any one or more of thesecharacteristics. Illustratively, the well-known terminologies“hydrophilic residues” relate to serine or threonine. “Hydrophobicresidues” refer to leucine, isoleucine, phenylalanine, valine oralanine, or the like. “Positively charged residues” relate to lysine,arginine, ornithine, or histidine. “Negatively charged residues” referto aspartic acid or glutamic acid. Residues having “bulky side chains”refer to phenylalanine, tryptophan or tyrosine, or the like. A list ofillustrative conservative amino acid substitutions is given below (aminoacids are L-amino acids unless indicated with “D-”). The symbol pSrepresents phosphoserine.

For Amino Acid (3- and 1- Letter Codes) Replace With Alanine (Ala, A)D-Ala, Gly, 2-aminoisobutyrate, β-Ala, L-Cys, D-Cys Arginine (Arg, R)D-Arg, Lys, D-Lys, Orn D-Orn, Propylguanidine Asparagine (Asn, N) D-Asn,Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic Acid (Asp, D-Asp, D-Asn, Asn,Glu, D-Glu, Gln, D-Gln D) Cysteine (Cys, C) D-Cys, S-Me-Cys, Met, D-Met,Thr, D-Thr Glutamine (Glu, Q) D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-AspGlutamic Acid (Glu, D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, E) D-Gln,Carboxyglutamate, Butyrate Glycine (Gly, G) Ala, D-Ala, Pro, D-Pro, Aib,β-Ala Histidine (His, H) D-His, D-Asn, Asn, D-Gln, Gln, D-Lys, Lys,D-Arg, Arg Isoleucine (Ile, I) D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine (Leu, L) Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile, Norleucine,Tert-Leucine Lysine (Lys, K) D-Lys, Arg, D-Arg, Orn, D-Orn Methionine(Met, M) D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine (Phe, F) D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline(Pro, P) D-Pro Serine (Ser, S) D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-CysThreonine (Thr, T) D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-ValTryptophan (Trp, W) D-Trp, D-Tyr, Tyr, D-Phe, Phe Tyrosine (Tyr, Y)D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine (Val, V) D-Val, Leu,D-Leu, Ile, D-Ile, Met, D-Met

In one embodiment, conservative side chain substitutions, such as L-I-V,S-C-T, E-D, N-Q and K-R, can be used to obtain analogues of any of thesingle chain peptides embraced herein or their comprising sequences fromTAT and MYB, or from TAT, CREB and MYB.

Linkers optionally between any sequence in a single chain peptide of theinvention may comprise one or more amino acids or a non-amino acid. Inone embodiment the linker is a sequence of one to about 8 glycines. Inone embodiment the linker is two glycines (GG).

The single chain peptide of the invention may be one of those sequencesdescribed herein, or may have one amino acid changed among thosedescribed herein. In another embodiment, the peptide may have two oneamino acids changed among those described herein. In another embodiment,the peptide may have three one amino acids changed among those describedherein. In another embodiment, the peptide may have four one amino acidschanged among those described herein. In another embodiment, the peptidemay have five or more one amino acids changed among those describedherein. The foregoing number of changes may apply to the single chainpeptide or any one or more of the sequences from TAT or MYB, or fromTAT, CREB or MYB, contained therein. In one embodiment, the peptidesequence of TAT, MYB or CREB may have 98% sequence homology with arespective peptide described herein. In one embodiment, the peptide mayhave 95% sequence homology with a peptide described herein. In oneembodiment, the peptide may have 90% sequence homology with a peptidedescribed herein. In one embodiment, the peptide may have 85% sequencehomology with a peptide described herein. In one embodiment, the peptidemay have 80% sequence homology with a peptide described herein. In oneembodiment, the peptide may have 75% sequence homology with a peptidedescribed herein. In one embodiment, the peptide may have 70% sequencehomology with a peptide described herein. In the foregoing embodiments,the peptide retains one or more biological activities described herein,or one or more activities are enhanced.

In another embodiment, deletions, truncations, additions, or othermodifications of the single chain peptide may be made, that retain oreven enhance the biological activity. One or more amino acids may beadded, deleted, including truncation of one or both ends of either the“TAT” and “MYB” portions of the peptide in one embodiment, or in the“TAT”, “CREB” or “MYB” portions of the peptide in another embodiment.

In another embodiment, the peptide may comprise both L-amino acids andD-amino acids, such that the peptide retains or has even enhancedbiological activity.

Biological activity means any one or more of the activities of peptidesdescribed herein, such as but not limited to cell penetration,competition with MYB:CBP interaction, interfering with MYB:CBPinteraction, reducing downstream activity from MYB:CBP interaction,downregulation of MYB-regulated genes, downregulation of MYC,downregulation of BCL2, inducing apoptosis in leukemic cells in vitro,anti-leukemic activity in vivo, and any combination of any of theforegoing.

In one embodiment, the portion of the cell penetrating peptide sequencefrom HIV-TAT in the single chain peptide of the invention comprises theL-amino acids YGRKKRRQRRR (SEQ ID NO:16). In a retro-inverso singlechain peptide or retro-inverso portion thereof, the sequence is theD-amino acids RRRQRRKKRGY (SEQ ID NO:17). In one embodiment, the portionof the MYB sequence that a single chain peptide of the inventioncomprises the L-amino acids KRIKELELLLMSTENELK (SEQ ID NO:18) which areamino acids 293-310 of human MYB. In a retro-inverso single chainpeptide, or a retro-inverso portion thereof, the sequence is the D-aminoacids KLENETSMLLLELEKIRK (SEQ ID NO:19). Corresponding MYB sequencesfrom other mammalian species are also embraced by this invention. Aswill be discussed below, in other embodiments, a fragment of either thecell penetrating peptide sequence from TAT, a fragment of the CBPbinding peptide sequence from MYB, or both, may be present in thesingle-chain peptide of the invention. Fragment refers to a contiguousportion of the full-length sequences, truncated at one or both ends. Thefragment retains the biological activity of the full peptide sequence.

In one embodiment, the portion of the cell penetrating peptide sequencefrom HIV-TAT in the single chain peptide of the invention comprises theL-amino acids YGRKKRRQRRR (SEQ ID NO:16). In a retro-inverso singlechain peptide or retro-inverso portion thereof, the sequence is theD-amino acids RRRQRRKKRGY (SEQ ID NO:17). In one embodiment, the portionof the MYB sequence that a single chain peptide of the inventioncomprises the L-amino acids LELLLMSTENELK (SEQ ID NO:21) which are aminoacids 298-310 of human MYB. In a retro-inverso single chain peptide, ora retro-inverso portion thereof, the sequence is the D-amino acidsKLENETSMLLLEL (SEQ ID NO:24). In one embodiment a linker, such as GG,may be disposed between the sequences. In one embodiment, the N-terminalamino acid may have an acetyl group, the C-terminus have an amide group,or both. Thus, the single chain, L-amino acid peptideYGRKKRRQRRGGLELLLMSTENELK (SEQ ID NO:38) and the single chain D-aminoacid peptide LELLLMSTENELKGGRRRQRRKKRGY (SEQ ID NO:39), and theirN-acetyl and C-amide analogues, are embraced herein.

In one embodiment, SEQ ID NO:1 (which also may be referred to herein asMYBMIM) and SEQ ID NO:2 (which also may be referred to herein as TATMYB)were developed in order to interfere with the assembly of the molecularMYB:CBP complex at micromolar concentrations and rapidly accumulate inthe nuclei of AML cells. As will be seen in the examples herein,treatment of AML cells with SEQ ID NO:1, but not with its inactivenear-isosteric analogue SEQ ID NO:3 (which also may be referred toherein as TG3), led to the displacement and dissociation of MYB:CBPcomplex in cells, causing rapid downregulation of MYB-dependent geneexpression including MYC and BCL2 oncogenes. As also will be seen in theexamples below, this was associated with obliteration of H3K27Ac-drivenoncogenic enhancers induced by CBP and enriched for MYB binding sites.Both human MLL-rearranged and non-rearranged AML cells, but not normalCD34+ umbilical cord blood progenitor cells, underwent sustainedmitochondrial apoptosis in response to SEQ ID NO:1 treatment, an effectthat could be partially blocked by ectopic expression of BCL2. Treatmentusing 50 mg/kg/day SEQ ID NO:1 impeded leukemia progression and extendedsurvival of immunodeficient mice engrafted with primary patient-derivedMLL-rearranged leukemia cells. These findings demonstrate the dependenceof human AML on MYB:CBP transcriptional dysregulation, and establishes apharmacologic approach for its therapeutic blockade following theteachings herein.

In one embodiment, an MYB site was identified amenable to therapeuticblockade using cell permeable peptidomimetic molecules. In oneembodiment, a cell permeable peptidomimetic inhibitor of the MYB:CBPinterface combines the HIV-TAT protein transduction domain with theinteracting domain of MYB to CBP. The peptide may be made in aretro-inverso conformation with D-amino acids with the goal ofmaintaining helical geometry of the peptide structure while protectingit from intracellular proteolysis. The activity of this peptide wascompared to that of a variant of the peptide (SEQ ID NO:3) whichincludes three glycine amino acid substitutions at the MYB 294, 302, and308 positions, thus substituting a small chain amino acid at the threesites in MYB that create the important charged (294, 308 positions) andhydrophobic (302) interactions between MYB and CBP. The SEQ ID NO:3peptide is ultimately unable to interact with CBP and as a consequence,is rendered inactive.

Thus, in one embodiment, peptides comprising L-amino acids or D-aminoacids (the latter typically in a retro-inverso configuration of thecorresponding L-amino acid sequence) are provided that interact with andreduce or prevent downstream MYB:CBP activity, downregulateMBY-regulated genes and kill leukemic cells in vivo. In one embodiment,the peptide consists of D-amino acids and isacetyl-KLENETSMLLLELEKIRKGGRRRQRRKKRGY-NH2 (SEQ ID NO:1). In anotherembodiment the peptide consists of L-amino acids and isacetyl-YGRKKRRQRRRGGKRIKELELLLMSTENELK-NH2 SEQ ID NO:2). In oneembodiment, the peptide consisting of D-amino acids isKLENETSMLLLELEKIRKGGRRRQRRKKRGY (SEQ ID NO:7). In another embodiment thepeptide consisting of L-amino acids is YGRKKRRQRRRGGKRIKELELLLMSTENELKSEQ ID NO:8). In other embodiments, the peptide may be modified at theamino terminal or carboxy terminal ends. In one embodiment, the aminoterminal end is acetylated. In another embodiment the carboxy terminalend is an amide. As noted above, both SEQ ID NO:1 and SEQ ID NO:2 havean amino terminal acetyl group and a carboxy terminal amide.

Various modifications of these peptides may be made for studying thecell penetration and other biological activities of the peptides. In oneembodiment, an inactive form of SEQ ID NO:1 is provided wherein glycinesare present at the 294, 302 and 308 positions of the MYB sequencetherein, providing the D-amino acid peptide[Ac]-KLGNETSMGLLELEKIGKGG-RRRQRRKKRGY-[NH2] (SEQ ID NO:3). In anotherembodiment, a biotin moiety is coupled to the amino terminal portion ofSEQ ID NO:2, providing D-amino acid peptideBiotin-YGRKKRRQRRRGGKRIKELELLLMSTENELK-NH2 (SEQ ID NO:4; may be referredto herein as BIOMYB). In another embodiment a biotin moiety is coupledto the carboxy terminal portion of SEQ ID NO:1 via a carboxy terminallysine, which is also an amide, providing D-amino acidsacetyl-KLENETSMLLLELEKIRKGGRRRQRRKKRGYK-biotin-NH₂ (SEQ ID NO:5; may bereferred to herein as RI-BIOMYB). In other experiments, afluorescein-conjugated TAT peptide is provided, using fluoresceinisothiocyanate and aminohexanoic acid at the amino terminus and amide atthe carboxy terminus, L-amino acids FITC-AHA-YGRKKRRQRRR-NH2, SEQ IDNO:6 (may be referred to herein as FITC-TAT; FITC representingfluorescein isothiocyanate and AHA representing aminohexanoic acid, thefluorophore and linker moieties, respectively). In another embodiment afluorescein-conjugated retro-inverso MYB peptide is provided, D-aminoacids FITC-AHA-KLENETSMLLLELEKIRK-NH₂ (SEQ ID NO:9).

As noted above, various modifications of the aforementioned peptides maybe carried out while preserving the biological activity of the peptidefor its intended purposes. By way of non-limiting example, the peptidesSEQ ID NO:2 and 8, which are comprised of L-amino acids, may be providedin retro-inverso configurations to protect against proteolyticdegradation in vivo; in such a configuration, D-amino acids are used andthe sequence is inverted, thus providing the identical orientation ofthe pendant side groups of the amino acids, SEQ ID Nos: 1 and 7,respectively. Chemical modifications of the amino and carboxy terminiare embodied herein, such as but not limited to, acetylation at the Nterminus, amide formation at the C terminus, or both.

In one embodiment, the modifications described for the single-chainpeptide comprising “TAT” and “MYB” above are applicable to a singlechain peptide comprising “TAT”, “CREB” and “MYB”, as described furtherdescribed below.

In one embodiment, the invention is directed to a single chain peptidecomprising a cell penetrating peptide sequence from HIV-TAT, a CBPbinding peptide sequence from CREB, and a CBP binding peptide sequencefrom MYB. In one embodiment, the sequence in the single chain peptidefrom HIV-TAT is L-amino acid sequence YGRKKRRQRRR (SEQ ID NO:16). In oneembodiment, the sequence of the CBP binding peptide sequence from CREBis the L-amino acid sequence RREILSRRPpSYRK (SEQ ID NO:20), and thesequence in the single chain peptide from MYB is L-amino acid sequenceLELLLMSTENELK (SEQ ID NO:21). In one embodiment, the cell penetratingpeptide sequence from HIV-TAT and the CBP binding peptide sequence fromCREB are joined by a linker. In one embodiment, the linker is GG. TheCBP binding peptide from MYB and the CBP binding peptide from CREB mayalso be joined by a linker.

In one embodiment, the cell penetrating peptide sequence from HIV-TAT,the CBP binding protein sequence from CREB and the CBP binding peptidesequence from MYB in the single chain peptide are composed of L-aminoacids. In one embodiment the N-to-C order of sequences is TAT-CREB-MYB.In one embodiment, the single chain peptide has the L-amino acidsequence YGRKKRRQRRRGGRREILSRRPpSYRKLELLLMSTENELK (SEQ ID NO:22).

In one embodiment, the cell penetrating peptide sequence from HIV-TAT,the CBP binding protein sequence from CREB, and the CBP binding peptidesequence from MYB are composed of D-amino acids, and the order of aminoacids in each sequence is inverted from that of the L-amino acidsequence (i.e., retro-inverso). In one embodiment, the sequence fromHIV-TAT is the D-amino acid sequence RRRQRRKKRGY (SEQ ID NO:17). In oneembodiment the sequence from CREB is the D-amino acid sequenceKRYpSPRRSLIERR (SEQ ID NO:23). In one embodiment, the sequence from MYBis the D-amino acid sequence KLENETSMLLLEL (SEQ ID NO:24). In oneembodiment, the N-to-C order of the retro-inverso sequences isMYB-CREB-TAT. In one embodiment, the single chain peptide has theD-amino acid sequence KLENETSMLLLELKRYpSPRRSLIERRGGRRRQRRKKRGY (SEQ IDNO:25).

In one embodiment, each of the cell penetrating peptide sequence fromHIV-TAT, the CBP binding peptide sequence from CREB, and the CBP bindingpeptide sequence from MYB peptide, or two or three of these sequenceswithin the single chain peptide, comprises one or more amino acidadditions, deletions or substitutions, or the N-terminus is acetylated,the C-terminus is amidated, or any combination thereof. Substitutionsinclude conservative or non-conservative substitutions. In oneembodiment, each of the cell penetrating peptide sequence from HIV-TAT,the CBP binding peptide sequence from CREB, the CBP binding peptidesequence from MYB, or two or three of these sequences within the singlechain peptide independently has at least 98%, at least 95%, at least90%, at least 85%, at least 80% or at least 75% sequence identity withthe respective fragment of the HIV-TAT, CREB, and MYB sequences withinSEQ ID NOS: 16, 20 and 18, respectively. Furthermore, the single chainpeptide of this embodiment may comprise longer or shorter fragments ofthe three respective components, as further described below. Theaforementioned embodiments apply to either the L-amino acid or D-aminoacid/reversed sequences of the invention.

In another embodiment, the cell penetrating peptide sequence from theTAT component of the single-chain peptide may be any fragment of L-aminoacids (SEQ ID NO:28). In another embodiment, the CREB component of thesingle-chain peptide may be any fragment of L-amino acids 124-146 ofCREB, RREILSRRPpSYRKILNDLSSDAP (SEQ ID NO:29). In one embodiment, thefragment of the CBP binding peptide sequence of CREB is L-amino acids133-146, pSYRKILNDLSSDAP (SEQ ID NO:30). In one embodiment, the fragmentof the CBP binding peptide sequence of CREB is L-amino acids 133-139,pSYRKILN (SEQ ID NO: 37). In one embodiment, the CBP peptide bindingfragment of MYB is L-amino acids 293-310, KRIKELELLLMSTENELK (SEQ IDNO:18). In another embodiment, the fragment of CBP binding peptide ofMYB is L-amino acids 298-310, LELLMSTENELK (SEQ ID NO:21). In anotherembodiment the fragment of MYB is L-amino acids 302-309, LMSTENEL (SEQID NO:31). In another embodiment, conservative side chain substitutions,such as L-I-V, S-C-T, E-D, N-Q and K-R can be used to obtain analogues.

If the component of the above fragment is in the D-amino acid andreversed order (retro inverso format), the corresponding peptides are:cell penetrating peptide from TAT component of the single-chain peptidemay be any fragment of D-amino acids (SEQ ID NO:17). In anotherembodiment, the CBP binding peptide from CREB component of thesingle-chain peptide may be any fragment of D-amino acids 124-146 ofCREB, PADSSLDNLIKRYpSPRRSLIERR (SEQ ID NO:33). In one embodiment, thefragment of CREB is D-amino acids 133-146, PADSSLDNLIKRYpS (SEQ IDNO:34). In one embodiment, the fragment of CREB is D-amino acids133-139, NLIKRYpS (SEQ ID NO:36). In one embodiment, the fragment of MYBis D-amino acids 293-310, KLENETSMLLLELEKIRK (SEQ ID NO:19). In anotherembodiment the fragment of MYB is D-amino acids 302-309, LENETSML (SEQID NO:35). As noted above, fragment refers to a continuous sequence ofamino acids from within the peptide, which can be truncated at eitherthe N- or C-terminus or both, and retains the desired activity of thepeptide. Furthermore, as noted above, the amino acid ranges denoted foreach sequence are that from the respective peptide in the L-amino acid,then provided as D-amino acids in the reverse order in the single chainpeptide or D-amino acid portion thereof. In another embodiment,conservative side chain substitutions, such as the D-amino acid isomersof L-I-V, S-C-T, E-D, N-Q and K-R can be used to obtain analogues.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids298-310 of the CBP binding peptide from MYB, L-amino acids LELLLMSTENELK(SEQ ID NO:21), and between them, amino acids 124-136 the CBP bindingpeptide sequence from CREB, L-amino acids RREILSRRPpSYRK (SEQ ID NO:20).In one embodiment, the sequences from HIV-TAT and from CREB are joinedby a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids293-310 of the CBP binding peptide from MYB, L-amino acidsKRIKELELLLMSTENELK (SEQ ID NO:18), and between them, amino acids 124-136the CBP binding peptide sequence from CREB, L-amino acids RREILSRRPpSYRK(SEQ ID NO:20). In one embodiment, the sequences from HIV-TAT and fromCREB are joined by a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids302-309 of the CBP binding peptide from MYB, L-amino acids LMSTENELK(SEQ ID NO:31), and between them, amino acids 124-136 the CBP bindingpeptide sequence from CREB, L-amino acids RREILSRRPpSYRK (SEQ ID NO:20).In one embodiment, the sequences from HIV-TAT and from CREB are joinedby a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids298-310 of the CBP binding peptide from MYB, L-amino acids LELLLMSTENELK(SEQ ID NO:21), and between them, amino acids 133-146 the CBP bindingpeptide sequence from CREB, L-amino acids pSYRKILNDLSSDAP (SEQ IDNO:30). In one embodiment, the sequences from HIV-TAT and from CREB arejoined by a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids302-309 of the CBP binding peptide from MYB, L-amino acids LMSTENELK(SEQ ID NO:31), and between them, amino acids 133-146 the CBP bindingpeptide sequence from CREB, L-amino acids pSYRKILNDLSSDAP (SEQ IDNO:30). In one embodiment, the sequences from HIV-TAT and from CREB arejoined by a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids293-310 of the CBP binding peptide from MYB, L-amino acidsKRIKELELLLMSTENELK (SEQ ID NO:18), and between them, amino acids 124-136the CBP binding peptide sequence from CREB, L-amino acids RREILSRRPpSYRK(SEQ ID NO:20). In one embodiment, the sequences from HIV-TAT and fromCREB are joined by a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids298-310 of the CBP binding peptide from MYB, L-amino acids LELLLMSTENELK(SEQ ID NO:21), and between them, amino acids 133-139 the CBP bindingpeptide sequence from CREB, L-amino acids pSYRKILN (SEQ ID NO:37). Inone embodiment, the sequences from HIV-TAT and from CREB are joined by alinker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids293-310 of the CBP binding peptide from MYB, L-amino acidsKRIKELELLLMSTENELK (SEQ ID NO:18), and between them, amino acids 133-139the CBP binding peptide sequence from CREB, L-amino acids pSYRKILN (SEQID NO:37). In one embodiment, the sequences from HIV-TAT and from CREBare joined by a linker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of L-amino acids, the N-terminal portion is amino acids 47-57of the cell penetrating peptide sequence from HIV-TAT, L-amino acidsYGRKKRRQRRR (SEQ ID NO:16); the C-terminal portion is amino acids302-309 of the CBP binding peptide from MYB, L-amino acids LMSTENELK(SEQ ID NO:31), and between them, amino acids 133-139 the CBP bindingpeptide sequence from CREB, L-amino acids pSYRKILN (SEQ ID NO:37). Inone embodiment, the sequences from HIV-TAT and from CREB are joined by alinker. In one embodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 298-310 of the CBP binding peptidefrom MYB, D-amino acids KLENETSMLLLEL (SEQ ID NO:24), the C-terminalportion is amino acids 47-57 of the cell penetrating peptide sequencefrom HIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); and betweenthem, amino acids 124-136 the CBP binding peptide sequence from CREB,D-amino acids KRYpSPRRSLIERR (SEQ ID NO:23). In one embodiment, thesequences from HIV-TAT and from CREB are joined by a linker. In oneembodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 293-310 of the CBP binding peptidefrom MYB, D-amino acids KLENETSMLLLELEKIRK (SEQ ID NO:19), theC-terminal portion is amino acids 47-57 of the cell penetrating peptidesequence from HIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); andbetween them, amino acids 133-146 the CBP binding peptide sequence fromCREB, D-amino acids PADSSLDNLIKRYpS (SEQ ID NO:34). In one embodiment,the sequences from HIV-TAT and from CREB are joined by a linker. In oneembodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 293-310 of the CBP binding peptidefrom MYB, D-amino acids KLENETSMLLLELEKIRK (SEQ ID NO:19), theC-terminal portion is amino acids 47-57 of the cell penetrating peptidesequence from HIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); andbetween them, amino acids 124-146 the CBP binding peptide sequence fromCREB, D-amino acids KRYpSPRRSLIERR (SEQ ID NO:23). In one embodiment,the sequences from HIV-TAT and from CREB are joined by a linker. In oneembodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 302-309 of the CBP binding peptidefrom MYB, D-amino acids LENETSML (SEQ ID NO:35), the C-terminal portionis amino acids 47-57 of the cell penetrating peptide sequence fromHIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); and between them,amino acids 124-136 the CBP binding peptide sequence from CREB, D-aminoacids KRYpSPRRSLIERR (SEQ ID NO:23). In one embodiment, the sequencesfrom HIV-TAT and from CREB are joined by a linker. In one embodiment thelinker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 298-310 of the CBP binding peptidefrom MYB, D-amino acids KLENETSMLLLEL (SEQ ID NO:24), the C-terminalportion is amino acids 47-57 of the cell penetrating peptide sequencefrom HIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); and betweenthem, amino acids 133-146 the CBP binding peptide sequence from CREB,D-amino acids PADSSLDNLIKRYpS (SEQ ID NO:34). In one embodiment, thesequences from HIV-TAT and from CREB are joined by a linker. In oneembodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 302-309 of the CBP binding peptidefrom MYB, D-amino acids LENETSML (SEQ ID NO:35), the C-terminal portionis amino acids 47-57 of the cell penetrating peptide sequence fromHIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); and between them,amino acids 133-146 the CBP binding peptide sequence from CREB, D-aminoacids PADSSLDNLIKRYpS (SEQ ID NO:34). In one embodiment, the sequencesfrom HIV-TAT and from CREB are joined by a linker. In one embodiment thelinker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 293-310 of the CBP binding peptidefrom MYB, D-amino acids KLENETSMLLLELEKIRK (SEQ ID NO:19), theC-terminal portion is amino acids 47-57 of the cell penetrating peptidesequence from HIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); andbetween them, amino acids 133-139 the CBP binding peptide sequence fromCREB, D-amino acids NLIKRYpS (SEQ ID NO:36). In one embodiment, thesequences from HIV-TAT and from CREB are joined by a linker. In oneembodiment the linker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 298-310 of the CBP binding peptidefrom MYB, D-amino acids KLENETSMLLLEL (SEQ ID NO:24), the C-terminalportion is amino acids 47-57 of the cell penetrating peptide sequencefrom HIV-TAT, D-amino acids RRRQRRKKRGY (SEQ ID NO:17); and betweenthem, amino acids 133-139 the CBP binding peptide sequence from CREB,D-amino acids NLIKRYpS (SEQ ID NO:36). In one embodiment, the sequencesfrom HIV-TAT and from CREB are joined by a linker. In one embodiment thelinker is GG.

Thus, in one embodiment, wherein the single chain peptide is comprisedentirely of D-amino acids, and the order of amino acids reversed fromthat in the corresponding L-amino acid sequence (i.e., retro-inverso),the N-terminal portion is amino acids 302-309 of the CBP binding peptidefrom MYB, D-amino acids LENETSML (SEQ ID NO:35), the C-terminal portionis the cell penetrating peptide sequence from HIV-TAT, D-amino acidsRRRQRRKKRGY (SEQ ID NO:17); and between them, amino acids 133-139 theCBP binding peptide sequence from CREB, D-amino acids NLIKRYpS (SEQ IDNO:36). In one embodiment, the sequences from HIV-TAT and from CREB arejoined by a linker. In one embodiment the linker is GG.

In one embodiment, the single chain peptide has the L-amino acidsequence is [Ac]-YGRKKRRQRRRGGRREILSRRPpSYRK-LELLLMSTENELK-[NH2] (SEQ IDNO:26) and in one embodiment, the single chain peptide has the D-aminoacid sequence [Ac]-KLENETSMLLLELKRYpSPRRSLIERRGGRRRQRRKKRGY-[NH2] (SEQID NO:27). In various embodiments, the N-terminus may be acetylated, theC-terminus amidated, or the combination of both.

In another embodiment nucleic acid molecules encoding any of thepeptides of the invention are provided, as well as vectors comprisingthe nucleic acid sequence of a peptide of the invention. In anotherembodiment, the method entails introduction of the genetic sequence thatencodes the peptides of this invention using, e.g., one or more nucleicacid delivery techniques. Nucleic acids of the invention include, inanother embodiment, DNA, RNA and mixtures of DNA and RNA, alone or inconjunction with non-nucleic acid components. In another embodiment, themethod comprises administering to the subject a vector comprising apolynucleotide sequence, which encodes a peptide of the presentinvention (Tindle, R. W. et al. Virology (1994) 200:54). In anotherembodiment, the method comprises administering to the subject naked DNAwhich encodes a peptide, or in another embodiment, two or more peptidesof this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). Eachpossibility represents a separate embodiment of the present invention.Moreover, nucleic acids encoding the single chain peptide may providefor facile synthesis or production of peptides for therapeutic use, byexpression in any number of biological systems.

Nucleic acids can be administered to a subject via any means as is knownin the art, including parenteral or intravenous administration, or inanother embodiment, by means of a gene gun. In another embodiment, thenucleic acids are administered in a composition, which correspond, inother embodiments, to any embodiment listed herein.

Vectors for use according to methods of this invention can comprise anyvector that facilitates or allows for the expression of a peptide ofthis invention. Vectors comprises, in some embodiments, attenuatedviruses, such as vaccinia or fowlpox, such as described in, e.g., U.S.Pat. No. 4,722,848, incorporated herein by reference. In anotherembodiment, the vector is BCG (Bacille Calmette Guerin), such asdescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vectors useful for therapeutic administration or immunizationof the peptides of the invention, e.g., Listeria, Salmonella typhivectors, and the like, will be apparent to those skilled in the art fromthe description herein.

Pharmaceutical Compositions

As discussed above, this invention provides novel compounds that havebiological properties useful for the treatment of any of a number ofconditions or diseases in which interfering with MYB:CBP interactionshave a therapeutically useful role.

The compounds and compositions of the invention may be administered to asubject or patients by any suitable route. Such routes include but arenot limited to parenterally, such as intravenously, subcutaneously,intradermally, intramucosally; topically; orally; intraocularly,intracamerally, or by inhalation. Depending on the location of thecancer to be treated, the compound or composition of the invention maybe delivered in or near the site.

Accordingly, in another aspect of the present invention, pharmaceuticalcompositions are provided, which comprise any one or more of thecompounds described herein (or a prodrug, pharmaceutically acceptablesalt or other pharmaceutically acceptable derivative thereof), andoptionally comprise a pharmaceutically acceptable carrier, diluent orexcipient. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents. Alternatively, acompound of this invention may be administered to a patient in needthereof in combination with the administration of one or more othertherapeutic agents. For example, additional therapeutic agents forconjoint administration or inclusion in a pharmaceutical compositionwith a compound of this invention may be an approved agent to treat thesame or related indication, or it may be any one of a number of agentsundergoing approval in the Food and Drug Administration that ultimatelyobtain approval for the treatment of any disorder related to cancer. Itwill also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a pro-drug or other adducts such as carrier proteins,fatty acids, dyes or polymers, or derivative of a compound of thisinvention which upon administration to a patient in need is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts of amines, carboxylic acids, and other types ofcompounds, are well known in the art. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting a free base or free acid function with a suitable reagent, asdescribed generally below. For example, a free base function can bereacted with a suitable acid. Furthermore, where the compounds of theinvention carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof may, include metal salts such as alkali metal salts, e.g.sodium or potassium salts; and alkaline earth metal salts, e.g. calciumor magnesium salts. Examples of pharmaceutically acceptable, nontoxicacid addition salts are salts of an amino group formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkalotic, alkenoic, cycloalkanoic and alkanedioic acids,in which each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, micro emulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut (peanut), corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose and starch. Such dosage forms may alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

In other embodiments solid dosage forms are provided. In certainembodiments, such solid dosage forms provide a higher than about a 20%oral bioavailability. As will be shown in the examples below, compoundsof the invention can be co-precipitated with one or more agents such asmannitol, a combination of mannitol and lacto bionic acid, a combinationof mannitol and gluconic acid, a combination of mannitol andmethanesulfonic acid, a combination of microcrystalline cellulose andoleic acid or a combination of pregelatinized starch and oleic acid. Theforegoing examples of one or more agents to aid in preparingformulations of inventive compound are merely illustrative andnon-limiting.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. Innon-limiting examples, one or more compounds of the invention may beformulated with at least one cytokine, growth factor or otherbiological, such as an interferon, e.g., alpha interferon, or with atleast another small molecule compound. Other non-limiting examplesinclude chemotherapeutic and other anti-cancer agents.

In certain embodiments, the pharmaceutical compositions of the presentinvention further comprise one or more additional therapeutically activeingredients (e.g., chemotherapeutic, anti-inflammatory and/orpalliative). For purposes of the invention, the term “Palliative” refersto treatment that is focused on the relief of symptoms of a diseaseand/or side effects of a therapeutic regimen, but is not curative. Forexample, palliative treatment encompasses painkillers, antinauseamedications and anti-sickness drugs.

Various embodiments of dosage ranges are contemplated by this invention.In one embodiment the dose amount is based on a weight of peptide perdose. In another embodiment the dose amount is based on a weight ofpeptide per kilogram of body weight of the patient. The dose amount maybe administered daily, twice a day, three times a day, four times a day,or more or less often. The abbreviation mg means milligram and theabbreviation mcg means microgram.

In one embodiment, the dosage is 10-20 mcg per dose. In anotherembodiment, the dosage is 20-30 mg per dose. In another embodiment, thedosage is 20-40 mcg per dose. In another embodiment, the dosage is 30-60mcg per dose. In another embodiment, the dosage is 40-80 mcg per dose.In another embodiment, the dosage is 50-100 mcg per dose. In anotherembodiment, the dosage is 50-150 mcg per dose. In another embodiment,the dosage is 100-200 mcg per dose. In another embodiment, the dosage is200-300 mcg per dose. In another embodiment, the dosage is 300-400 mcgper dose. In another embodiment, the dosage is 400-600 mcg per dose. Inanother embodiment, the dosage is 500-800 mcg per dose. In anotherembodiment, the dosage is 800-1000 mcg per dose.

In one embodiment, the dosage is 10-20 mg per dose. In anotherembodiment, the dosage is 20-30 mg per dose. In another embodiment, thedosage is 20-40 mg per dose. In another embodiment, the dosage is 30-60mg per dose. In another embodiment, the dosage is 40-80 mg per dose. Inanother embodiment, the dosage is 50-100 mg per dose. In anotherembodiment, the dosage is 50-150 mg per dose. In another embodiment, thedosage is 100-200 mg per dose. In another embodiment, the dosage is200-300 mg per dose. In another embodiment, the dosage is 300-400 mg perdose. In another embodiment, the dosage is 400-600 mg per dose. Inanother embodiment, the dosage is 500-800 mg per dose. In anotherembodiment, the dosage is 800-1000 mg per dose.

In one embodiment, the dosage is 10-20 mcg/kg per dose. In anotherembodiment, the dosage is 20-30 mcg/kg per dose. In another embodiment,the dosage is 20-40 mcg/kg per dose. In another embodiment, the dosageis 30-60 mcg/kg per dose. In another embodiment, the dosage is 40-80mcg/kg per dose. In another embodiment, the dosage is 50-100 mcg/kg perdose. In another embodiment, the dosage is 50-150 mcg/kg per dose. Inanother embodiment, the dosage is 100-200 mcg/kg per dose. In anotherembodiment, the dosage is 200-300 mcg/kg per dose. In anotherembodiment, the dosage is 300-400 mcg/kg per dose. In anotherembodiment, the dosage is 400-600 mcg/kg per dose. In anotherembodiment, the dosage is 500-800 mcg/kg per dose. In anotherembodiment, the dosage is 800-1000 mcg/kg per dose.

In one embodiment, the dosage is 10-20 mg/kg per dose. In anotherembodiment, the dosage is 20-30 mg/kg per dose. In another embodiment,the dosage is 20-40 mg/kg per dose. In another embodiment, the dosage is30-60 mg/kg per dose. In another embodiment, the dosage is 40-80 mg/kgper dose. In another embodiment, the dosage is 50-100 mg/kg per dose. Inanother embodiment, the dosage is 50-150 mg/kg per dose. In anotherembodiment, the dosage is 100-200 mg/kg per dose. In another embodiment,the dosage is 200-300 mg/kg per dose. In another embodiment, the dosageis 300-400 mg/kg per dose. In another embodiment, the dosage is 400-600mg/kg per dose. In another embodiment, the dosage is 500-800 mg/kg perdose. In another embodiment, the dosage is 800-1000 mg/kg per dose.

In another embodiment, the total amount of peptide per dose or per dayis one of the above amounts. In another embodiment, the total peptidedose per dose is one of the above amounts.

Each of the above doses represents a separate embodiment of the presentinvention.

Methods of Treatment

The single chain peptides embodied here are useful for the treatment ofcancer and other dysproliferative diseases. In one embodiment the canceris a hematopoietic cancer. In other embodiments, the cancer is, by wayof non-limiting example, T-cell acute lymphoblastic leukemia or acutelymphocytic leukemia (ALL), acute myeloid leukemia (AML), and chronicmyeloid leukemia (CML). Other cancers amenable to the compounds,compositions and uses here include but are not limited to small celllung cancer, renal cell carcinoma, adenoid cystic carcinoma, squamouscell carcinoma of the head and neck, neuroblastoma and pancreaticcancer. In another embodiment, any cancer in which the interaction ofMYB and CBP is pathogenetic is amenable to treatment using thecompounds, compositions and methods described herein.

In one embodiment the subject has cancer. In one embodiment, the subjectis at risk for developing cancer. In one embodiment, the subject is inremission from cancer. In other embodiments, the cancer is transformedfollicular lymphoma, mantel cell lymphoma, breast cancer, ovariancancer, hepatocellular carcinoma, and non-small cell lung cancer, aswell as gastric cancer, Ewing sarcoma and lung adenocarcinoma. These aremerely examples of cancers amenable to the methods and uses of theagents herein described.

In another embodiment, the cancer is a lymphoma. In another embodiment,the cancer is a desmoplastic small round cell tumor. In anotherembodiment, the cancer is a mesothelioma. In another embodiment, thecancer is a gastric cancer. In another embodiment, the cancer is a coloncancer. In another embodiment, the cancer is a lung cancer. In anotherembodiment, the cancer is a breast cancer. In another embodiment, thecancer is a germ cell tumor. In another embodiment, the cancer is anovarian cancer. In another embodiment, the cancer is a uterine cancer.In another embodiment, the cancer is a thyroid cancer. In anotherembodiment, the cancer is a hepatocellular carcinoma. In anotherembodiment, the cancer is a thyroid cancer. In another embodiment, thecancer is a liver cancer. In another embodiment, the cancer is a renalcancer. In another embodiment, the cancer is a Kaposi's sarcoma. Inanother embodiment, the cancer is a sarcoma. In another embodiment, thecancer is any other carcinoma or sarcoma.

In another embodiment, the cancer is a solid tumor. In anotherembodiment, the solid tumor is associated with a cancer. In anotherembodiment, the solid tumor is associated with a myelodysplasticsyndrome (MDS). In another embodiment, the solid tumor is associatedwith a non-small cell lung cancer (NSCLC). In another embodiment, thesolid tumor is associated with a lung cancer. In another embodiment, thesolid tumor is associated with a breast cancer. In another embodiment,the solid tumor is associated with a colorectal cancer. In anotherembodiment, the solid tumor is associated with a prostate cancer. Inanother embodiment, the solid tumor is associated with an ovariancancer. In another embodiment, the solid tumor is associated with arenal cancer. In another embodiment, the solid tumor is associated witha pancreatic cancer. In another embodiment, the solid tumor isassociated with a brain cancer. In another embodiment, the solid tumoris associated with a gastrointestinal cancer. In another embodiment, thesolid tumor is associated with a skin cancer. In another embodiment, thesolid tumor is associated with a melanoma.

Methods of Making Peptides

Methods for synthesizing peptides are well known in the art. In anotherembodiment, the peptides of this invention are synthesized using anappropriate solid-state synthetic procedure (see for example, Stewardand Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco,Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23:451). The activity of these peptides is tested, in other embodiments,using assays as described herein.

In another embodiment, the peptides of this invention are purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for protein purification.In another embodiment, immuno-affinity chromatography is used, wherebyan epitope is isolated by binding it to an affinity column comprisingantibodies that were raised against that peptide, or a related peptideof the invention, and were affixed to a stationary support.

Materials and Methods.

Cell culture. The human AML lines MV411, MOLM13, ML2, and HL60 wereobtained from the American Type Culture Collection (ATCC, Manassas, Va.,USA). Umbilical cord blood was obtained from the New York Blood Center.The identity of all cell lines was verified by STR analysis (GeneticaDNA Laboratories, Burlington, N.C., USA) and absence of Mycoplasma sp.contamination was determined using Lonza MycoAlert (Lonza Walkersville,Inc., Walkersville, Md., USA). Cell lines were cultured in 5% CO2 in ahumidified atmosphere at 37° C. in RPMI medium supplemented with 10%fetal bovine serum (1135) and antibiotics (100 U/ml penicillin and 100μg/ml streptomycin).

Molecular mechanics simulations. The solution NMR structure of KIXdomain of CBP bound to the transactivation domain of C-MYB (PDB code1SB0) was used a starting point for simulations of both L- and D-aminoacid MYB-CBP complexes. Specifically, the NMR structure with the lowestroot-mean-square-deviation (RMSD) from the average of the ensemble of 20solution NMR structures was selected (model 5). D-amino acid MYB peptidewas built with Simulaid program using the NMR structure ofprotein-peptide complex and converting C-MYB peptide from L-amino acidsto D-amino acids in the presence of CBP. Simulations were performedusing the Desmond molecular dynamics program. The starting structureswere solvated with 6615 and 6714 SPC water molecules, respectively, witha 5 Å buffer of water in a rectangular box. Three chloride ions wereadded to both systems to maintain electric neutrality. The OPLS3 forcefield was used to describe both L- and D-amino acid peptide-proteincomplexes. For each system a relaxation phase, with a combination ofBrownian dynamics and restrained molecular dynamics phases was performedto equilibrate the systems. Periodic boundary conditions with a cutoffof 0.9 nm for both particle mesh Ewald and Lennard-Jones interactionswere used. Each equilibrated system was then subjected to 60 nssimulations with identical parameters. Simulations were performed usingthe constant pressure and constant temperature (NPT) ensemble with aBerendsen thermostat and barostat. The equations of motion wereintegrated using RESPA with a time step of 2.0 fs for bonded andshort-range non-bonded interactions, and 6.0 fs for long-rangeelectrostatic interactions. System coordinates were saved every 5 ps.

Expression and purification of recombinant CBP KIX domain. BL21(DE3)cells (Invitrogen) transformed with pGEX-KIX plasmid were induced at 37°C. with isopropyl β-D-1-thiogalactopyranoside for 3 hours. Cells werelysed in 50 mM Tris-HCl pH 7.3, 150 mM NaCl, 0.1% TWEEN-20, 1 mM DTT, 5mM EDTA, supplemented with protease inhibitors described above andsonicated for ten minutes (15 sec on, 15 sec off, 40% amplitude) usingthe Misonix probe sonicator (Qsonica, Newtown, Conn.). Lysate wascleared by centrifugation for 1 h at 21,800×g at 4° C. Cleared lysatewas incubated with 4 mL glutathione agarose resin slurry (GoldBio) for 1h at 4° C. to capture GST-KIX. Resin was washed four times with 50 mMTris-HCl pH 7.4, 150 mM NaCl. KIX domain was cleaved from GST byincubation of resin-bound GST-KIX with 160 U thrombin (GE Healthcare)overnight at room temperature. Resin was centrifuged at 500×g for 5 min.Supernatant containing cleaved MX was collected and dialyzed at 4° C.against 50 mM MOPS pH 6.5, 50 mM NaCl, 10% glycerol, 1 μMtris-2-carboxyethylphosphine. MX was purified using a linear gradient of50 mM to 1 M NaCl by cation exchange chromatography using MonoS 5/50 GLcolumn (GE Healthcare). Fractions containing purified KIX were dialyzedagainst 50 mM potassium phosphate pH 5.5, 150 mM NaCl, 10 μMtris-2-carboxyethylphosphine, 30% glycerol, and stored at −80° C.

Microscale thermopheresis (MST). For binding affinity studies,interaction of purified recombinant KIX with FITC-conjugated peptideswas optimized and performed in MST buffer (50 mM Phosphate, 150 mM NaCl,0.01% NP-40, pH 5.5) using varying laser powers (FITC-MYB, 250 nM at LEDpower 40%, FITC-MYBMIM at 500 nM at LED power 50% and FITC-TAT at 500 nMat LED power of 50%) and a blue laser equipped Monolith NT.115(NanoTemper Technologies). Prior to each run, protein aggregation wasminimized by centrifuging the solutions at 15,000 rpm for 8 minutes.FITC-peptide (fixed concentration) was mixed with increasingconcentrations of KIX (0.015 to 50 μM) and loaded onto 16 Premium Coatedcapillaries. The MST measurements were taken at RT and a fixed IR-laserpower of 80% for 10 sec per capillary. GraphPad Prism was used to fitthe normalized data and determine apparent KD values, represented aspercent of fraction bound.

Confocal microscopy. Live confocal imaging was performed using the LeicaSP8 confocal microscope and 63× objective with 1 μm z-stack images.Cells were applied to a poly-L-lysine coated chambered Nunc Lab-tek IIslide and incubated for 2 hours at 37° C. Prior to imaging,FITC-conjugated peptides were added to cell suspensions at aconcentration of 50 nM. Cells were counter-stained using Hoechst 33342and Mitotracker Red CMX ROS (MProbes) for 10 minutes at a final dilutionof 1:10,000 prior to imaging.

Western blot analysis. Cells were lysed in RIPA buffer (Thermo Fisher)supplemented with a protease inhibitor mix comprised of AEBSF (0.5 mMconcentration, Santa Cruz, SC-202041B), Bestatin (0.01 mM concentration,Fisher/Alfa Aesar, J61106-MD), Leupeptin (0.1 mM concentration, SantaCruz, SC-295358B), and Pepstatin (0.001 mM concentration, Santa Cruz,SC-45036A). Lysates were mechanically disrupted using Covaris S220adaptive focused sonicator, according to the manufacturer's instructions(Covaris, Woburn, Calif.). Lysates were cleared by centrifugation for 15min at 18,000×g and clarified lysates were quantified using thebicinchoninic acid assay (Pierce). Clarified lysates (20 μg of protein)were resolved using sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, and electroeluted using the Immobilon FL PVDF membranes(Millipore, Billerica, Mass., USA). Membranes were blocked using theOdyssey Blocking buffer (Li-Cor, Lincoln, Nebr., USA). The followingprimary antibodies were used as indicated: anti-MYB (1:500, C-19, SantaCruz), anti-CBP (1:500, A-22, Santa Cruz), anti-n actin (1:1000,8H10D10, Cell Signaling). Blotted membranes were visualized usingsecondary antibodies conjugated to IRDye 800CW or IRDye 680RD (Goatanti-rabbit, 1:15,000, and goat anti-mouse, 1:15,000) and the OdysseyCLx fluorescence scanner, according to manufacturer's instructions(Li-Cor, Lincoln, Nebr., USA).

Co-immunoprecipitation analysis. 7.5 μg of indicated antibodies wereconjugated to 1 mg M-270 Epoxy-coated magnetic beads (Invitrogen)according to manufacturer's instructions. 1×10⁷ MV411 cells werecollected and washed in cold PBS. Washed cell pellets were resuspendedin 350 μL cold lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mMEDTA, 1 mM DTT, 0.5% Triton X-100, supplemented with protease inhibitorsdescribed above) and incubated on ice for 10 min. Cells weremechanically disrupted using the Covaris S220 adaptive focused sonicatorat 50 W peak power, 10% duty cycle, 200 cycles per burst at 4° C. for300 sec. Lysate was clarified by centrifugation for 15 min at 18,000×gat 4° C. Supernatant (cleared lysate) was diluted in 1.05 mL dilutionbuffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol,supplemented with protease inhibitors described above for a total volumeof 1.4 mL. Diluted lysate was added to 1 mg beads, andimmunoprecipitation proceeded for 3 h at 4° C. with rotation. Beads werethen washed with lysis buffer twice. Proteins were eluted in 20 μL EBbuffer (Invitrogen) for 5 min at room temperature, and eluate wasneutralized with 2 μL 1M Tris pH 11. Samples were prepared for Westernblot by addition of Laemmli buffer with 50 mM DTT and incubation at 95°C. for 5 min. Presence of MYB and CBP was identified by Western blot asdescribed above.

Streptavidin affinity purification. Streptavidin magnetic beads (Pierce)were washed with TBST twice prior to use. Biotinylated MYBMIM (L-aa orD-aa) was conjugated to 150 μL streptavidin bead slurry (1.5 mg beads,binding capacity 3500 pmol biotinylated fluorescein per mg) byincubation at room temperature for 2 h in 1 mL TBST. Peptide-conjugatedbeads were washed twice in 1 mL TBST. 1×10⁷ cells were collected andwashed in cold PBS. Washed cell pellets were lysed in 350 μL of 50 mMTris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, mM DTT, 1% octyl-β-glucoside,1% Pluronic F-supplemented with 4-(2-aminoethyl)benzenesulfonyl fluoridehydrochloride, bestatin, leupeptin, and pepstatin and incubated on icefor 10 min. Cells were mechanically disrupted using the Covaris S220adaptive focused sonicator at 50 W peak power, 10% duty cycle, 200cycles per burst at 4° C. for 300 sec. Lysate was clarified bycentrifugation for 15 min at 18,000×g at 4° C. Supernatant (clearedlysate) was diluted in 1.05 mL dilution buffer (50 mM Tris-HCl, 150 mMNaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, supplemented with proteaseinhibitors described above) for a total volume of 1.4 mL. TBST wasremoved from peptide-conjugated streptavidin bead slurry, and dilutedlysate was added to 1.5 mg beads. For competition experiments, 10-, 5-,or 2.5-fold excess non-biotinylated peptide was added to the lysate justprior to addition to streptavidin beads. Pulldown and peptidecompetition proceeded for 3 h at 4° C. with rotation. Beads were washedtwice with lysis buffer. Bound proteins were eluted by adding 40 μLLaemmli buffer with 50 mM DTT and incubated for 5 min at 95° C. Presenceof CBP was identified by Western blot as described above.

Chromatin immunoprecipitation and sequencing (ChIP-seq). Briefly, cellswere fixed in 1% formalin in phosphate-buffered saline (PBS) for 10minutes at room temperature. Glycine (125 mM final concentration) andTris-HCl pH 8 (100 mM final concentration) was added to the cells andcells were washed twice in ice-cold PBS and resuspended in sodiumdodecyl sulfate (SDS) lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl,pH 8.1). Lysates were sonicated using the Covaris S220 adaptive focusedsonicator to obtain 100-500 bp chromatin fragments (Covaris, Woburn,Calif.). Lysates containing sheared chromatin fragments were resuspendedin 0.01% SDS, 1.1% Triton-X100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1,167 mM NaCl. Lysates and antibody-coupled beads were incubated overnight at 4° C. Precipitates were washed sequentially with Mixed MicelleWash Buffer (15 ml 5M NaCl-150 mM Final, 10 ml 1M Tris-Cl pH 8.1, 5 ml0.5M EDTA, pH 8.0, 40 ml 65% w/v sucrose, 1 ml 10% NaN3, 25 ml 20%Triton X-100, 10 ml 10% SDS, Add dH2O to 500 ml), LiCl washing solution(0.5% deoxycholic acid, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 10 mMTris-Cl pH 8.0, 0.2% NaN3) and then TBS buffer (20 mM Tris-Cl pH 7.4,150 mM NaCl). Elution performed in elution buffer (1% SDS, 0.1 MNaHCO3). ChIP-seq libraries were generated using the NEBNext ChIP-seqlibrary prep kit following the manufacturer's protocol (New EnglandBiolabs, Ipswich, Mass., USA). Libraries were sequenced on the llluminaHiSeq 2500 instruments, with 30 million 2×50 bp paired reads.

Catalog Antibody Manufacturer number Lot Dilution H3K27ac Abcam 4729GR200563-2 1:100 H3K4me1 Abcam 8895 GR114265-2 1:100 H3K4me3 CellSignaling 9751 7 1:100 Technologies H3K79me2 Cell Signaling 5427 4 1:100Technologies H3K36me3 Abcam 9050 GR166781-1 1:100 H3K27me3 Millipore07-449 2736613 1:100 CBP SantaCruz sc-369, A-22 L2815 1:10 MYB Abcamab45150 1:200

Cell viability analysis. Cells were resuspended and plated at aconcentration of 2×105 cells in 200 μL in 96-well tissue culture plates.Media with peptides was replaced every 48 hours. To assess the number ofviable cells, cells were resuspended in PBS and 10 μL mixed in a 1:1ratio with 0.4% Trypan Blue (Thermo Fisher) and counted using ahematocytometer (Hausser Scientific, Horsham, Pa., USA). To assessviability using an ATP-based assay, cell viability was assessed usingthe CellTiter-Glo Luminescent Viability assay, according to themanufacturer's instructions (Promega). Luminescence was recorded usingthe Infinite M1000Pro plate reader using integration time of 250milliseconds (Tecan).

Flow cytometric analysis of Apoptosis. Cells were resuspended to aconcentration of 1×106 cells were plated in triplicate in a 12-welltissue culture plate. For assessment of annexin V staining, cells werewashed with PBS and then resuspended in PBS with Annexin V-APC(BioLegend) and propidium iodide at a dilution of 1:1000. Forintracellular detection of cleaved caspase 3, cells were fixed andpermeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilizationsolution according to the manufacturer's instructions (BD Biosciences.Cells were then stained using the Alexa Fluor 647-conjugated anti-activecaspase-3 (BD Biosciences) at a dilution of 1:50. Cells were incubatedfor 30 minutes room temperature in the dark, washed, and then analyzedusing the BD LSRFortessa cell analyzer.

Giemsa staining of cells for Morphology. MOLM13 cells were resupended toa concentration of 1×106 cells in 1 milliliter of PBS. Using thebenchtop Cytospin Centrifuge instrument (ThermoFisher Scientific), 200uL of the cell suspension was applied white clipped Cytofunnels(ThermoFisher Scientific) to glass microscope slides (2×105cells/slide). Dip Quick Stain (J-322, Jorgensen Laboratories, Inc) wasused for per manufacturer's protocol for the polychromic stain of cells.

Quantitative RT-PCR. RNA was isolated using Trizol reagent according tothe manufacturer's instructions (Life Technologies). Complementary DNAwas synthesized using the SuperScript III First-Strand Synthesis systemaccording to the manufacturer's instructions (Invitrogen). Quantitativereal-time PCR was performed using the KAPA SYBR FAST PCR polymerase with20 ng template and 200 nM primers, according to the manufacturer'sinstructions (Kapa Biosystems, Wilmington, Mass., USA). PCR primers arelisted below. Ct values were calculated using ROX normalization usingthe ViiA 7 software (Applied Biosystems).

Primer Sequence, 5′→3′ GAPDH, forwardAATCCCATCACCATCTTCCA (SEQ ID NO: 10) GAPDH, reverseTGGACTCCACGACGTACTCA (SEQ ID NO: 11) BCL2, forwardCTGCACCTGACGCCCTTCACC (SEQ ID NO: 12) BCL2, reverseCACATGACCCCACCGAACTCAAAGA (SEQ ID NO: 13) MYC, forwardTTCCCCTACCCTCTCAACGACAG (SEQ ID NO: 14) MYC, reverseCCTCATCTTCTTGTTCCTCCTCAG (SEQ ID NO: 15)

Retrovirus production and cell transduction. The MIG-BCL2 vector waspackaged using pUMVc and pCMV-VSVG vectors in HEK 293T cells and theFuGENE 6 transfection reagent, according to manufacturer's instructions(Promega). Virus supernatant was collected at 48 and 72 hourspost-transfection, pooled, filtered and stored at −80° C. Cells weretransduced with virus particles at a multiplicity of infection of 1 byspin inoculation for 90 minutes at 3500 rpm at 35° C. in the presence of8 μg/ml hexadimethrine bromide. Two days after transduction, cells wereisolated using fluorescence-activated cell sorting (FACSAria III, BDBioscience, San Jose, Calif., USA).

Blood progenitor colony forming assays. Mononuclear cells were isolatedfrom cord blood using Ficoll-Paque PLUS density centrifugation andenriched for CD34+ cells using the CD34 MicroBead Kit UltraPure,according to the manufacturer's instructions (Miltenyi Biotech). CD34+cells were resuspended to a concentration of 1×105 cells/mL. MethocultH4303 Optimum (Stemcell Technologies, Catalog no. 04034 with FBS, BSAand recombinant cytokines rhSCF, rhGM-CSF, rhG-CSF, rhIL3, andrhErythropoietin) semi-solid media was used for the growth ofhematopoietic progenitor cells in colony-forming units. Methocult andCD34+ cells were mixed in a ratio of 1:10 (cells:Methocult) for a finalcell concentration plated of 1000 cells/dish. TG3 or MYBMIM peptide wereadded to this solution for a final concentration of 20 □M. Mixture wasvortexed for 30 seconds and incubated at room temperature for 5 minutes.Using a blunt end 18 G needle, 1.1 mL of the solution was added to a35×10 mm dish and then tilted to provide even coverage on plate. Peptidetreatment conditions were plated in biological triplicates. 35×10 mmdishes placed into a larger 100×15 mm dish with 1 35×10 mm dish filledwith sterile H20). Dishes were incubated at 37° C. with 5% CO2 for 14days. Both erythroid progenitor and granulocyte-macrophage progenitorswere observed and quantified.

Mouse studies. All mouse experiments were carried out in accordance withinstitutional animal protocols. Two hundred thousand primary AMLMLL-rearranged leukemia cells were suspended in 200 ml of PBS andtransplanted via tail vein injection into 8-week-old sublethallyirradiation (200 rad) female NOD.Cg-Prkdc(scid)Il2rg(tm1Wj1/SzJ mice(The Jackson Laboratory, Bar Harbor, Me., USA). Recipient mice weremaintained on antibiotic supplementation in chow (0.025% trimethoprim,0.124% sulfamethoxazole, Sulfatrim). Three days after transplant, micewere randomly assigned to experimental treatment groups. MYBMIM peptidesuspended in PBS was administered daily through intraperitonealinjection at a daily dose of 50 mg/kg. Mice were treated from days 3-17of this study for a total of 14 days and then monitored daily withclinical examination for survival analysis.

Statistical Analysis. For comparisons between two sample sets,statistical analysis of means was performed using 2-tailed, unpairedStudent's t-tests. Survival analysis was done using the Kaplan-Meiermethod, as assessed using a log-rank test. For gene expression analysis,statistical significance was assessed using paired t-tests.

Example 1. Peptide Competes with the MYB:CBP Complex in AML

Through molecular modeling, there is in vitro binding of engineeredL-amino acid (L-aa; SEQ ID NO:2) and D-amino acid (Daa; SEQ ID NO:1)containing peptides to CBP (FIG. 1a ). Both Laa and Daa peptides wereconjugated to FITC by the Tufts University Core Facility. Throughplasmid DNA isolation from a pGEX backbone, the MX domain of CBP waspurified and utilized to determine binding affinity to bothFITC-conjugated peptides. Using the NanoTemper NT115 instrument, bindingaffinity was established in a microscale thermopheresis binding assay(FIG. 1b ). The rapid and efficient intracellular penetration of aFITC-conjugated peptide (SEQ ID NO:6) in a human AML cell line usingconfocal microscopy was demonstrated. There is intranuclear penetrationof the FITC-conjugated peptide after incubation with 50 nM concentrationof peptide for 20 minutes indicated by the FITC-Hoechst overlay (FIG. 1c). In a biochemical pull-down assay, using biotinylated MYBMIM (SEQ IDNO:5), streptavidin coated beads were treated with BIOMYB (SEQ ID NO:4)and with AML cell lysates and were able to pull down CBP. This showsthat there is specific binding of the MYBMIM peptide (SEQ ID NO:1) toits target CBP (FIG. 1d ). In showing the binding and intracellularpenetration of these peptides, biochemical assays here support thatMYBMIM (SEQ ID NO:1) is competing in a specific manner with the MYB:CBPcomplex in AML cells.

Detailed description of FIG. 1: MYBMIM competes with the MYB:CBP complexin AML. (a) Molecular Dynamic Simulation modeling was performed tocompare the molecular interactions within the native MYB:CBP complex tothe interaction between a MYB peptide to CBP (L-amino acid peptide,labeled as L-aa (SEQ ID NO:2), and D-amino acid, labeled MYBMIM, SEQ IDNO:1, are compared here). There was no significant difference in theconformation of MYB:CBP complex structures. (b) MicroscaleThermopheresis (MST) was performed here to analyze the binding affinityof MYB peptides to the purified CBP-KIX domain. FITC-MYB (L-aa peptide,green line, KD=5.25+0.6 μM) interacts with purified KIX at a higherbinding affinity than FITC-MYBMIM (D-aa peptide, red line, KD=25.4+2.3μM). There is no interaction seen between FITC-TAT and KIX (black line),n=3 for each curve. (c) The intranuclear penetration of FITC-labelledTAT peptides in MOLM13 cells is shown here using confocal microscopy. InFITC-TAT treated cells, there is an overlap of FITC and Hoechst in thenucleus as compared to control, untreated, cells. One micron z-stackimages were obtained using a 63× objective and 2× zoom with a singlestack image of maximum projection shown here (scale bar indicates 10microns). (d) Western blot showing specific binding ofbiotinylated-MYBMIM to CBP in MV411 human AML cells. BIOMYB indicatesbiotinylated MYBMIM containing L-aa and RI-BIOMYB (SEQ ID NO:5)indicates biotinylated MYBMIM containing D-aa. + or − symbols indicatethe presence or absence of respective non-biotinylated peptide, showingspecific competition of CBP binding in cells.

Example 2. Peptide Downregulates MYB-Regulated Genes and InducesApoptosis

Treatment of human AML cell lines with MYBMIM in vitro reveals aninduction of AML cell death that is seen in MOLM13, MV411, ML2, and HL60cell lines in a dose-dependent effect with MYBMIM, 10 μM and 20 μMdoses, as compared to its inactive isostere TG3, 10 μM and 20 μM doses(FIG. 2a ). Flow cytometric analysis of apoptosis using Annexin-V APCstaining in MV411 cells in vitro with MYBMIM treatment at 20 μM dose for24 hours (FIG. 2b ) reveals an induction of apoptosis. Morphologicalchanges are evident in Wright-Giemsa stains of cytospun MV411 cells withcell death seen at 40× immersion oil microscopy (FIG. 2c ). In trying toidentify the downstream gene expression effects of MYBMIM in human AMLcells, human AML cells were treated for six hours with the MYBMIMpeptide, using both TG3 inactive peptide and control untreated cells asnegative controls. RNA-seq data reveals significant downregulation ofMYB-dependent gene expression in MYBMIM treatment vs TG3 and control(FIGS. 2d ) and a volcano plot (FIG. 2e ) comparing the log-fold changevs p-adjusted values reveals significantly downregulated genes BCL2 andMYC, which are highlighted. To confirm this pattern of MYB-dependentdownregulation, RT-qPCR was performed to show downregulation of MYC andBCL2 mRNA expression in human AML cell lines as a percentage of GAPDH,(FIG. 2f ). Ectopic expression of Bcl2 in MV411 cells reveals a partialrescue of the MYBMIM induced apoptosis as measured in a cell viabilityassay using the CellTiter Glo reagent kit, (FIG. 2g ). There isreproducible downregulation of MYB target genes with a specific effectof AML cell death that is mediated through apoptosis.

Detailed description of FIG. 2: MYBMIM downregulates MYB-regulated genesand induces apoptosis. (a) Quantification of live AML cells using Trypanblue exclusion after MYBMIM treatment. Live MOLM13, MV411, ML2, and HL60cells were counted on Day 6 using a hemocytometer. Peptide treatment wasapplied directly to cell culture with inactive TG3 (dosed at 10 μM and20 μM) and MYBMIM (dosed at 10 μM and 20 μM) every 48 hours for a 6 dayperiod. Cells were plated and treated in biological triplicates withsymbols indicating p<0.05 (*p=0.0046, **p=0.0011, {circumflex over( )}p=0.00001, {circumflex over ( )}{circumflex over ( )}p=0.0004,#p=0.05, ##p=0.0039, +p=0.00016, ++p=0.0013). Each set of barsrepresents, from left to right, control, TG3 at 10 μM, TG3 at 20 μM,MYBMIM at 10 μM, MYBMIM at 20 μM. (b) Induction of apoptosis observedwith MYBMIM treatment of human AML cells. Flow cytometric analysis ofAnnexin-V and DAPI staining of MOLM13 cells after treatment with TG3 (20μM) and MYBMIM (20 μM) for 24 hours. % indicating percentage of cellsthat are both Annexin and DAPI positive. (c) MYBMIM induces cell deathwithout morphologic evidence of differentiation. Representative Giemsastained images of MOLM13 cells after treatment with TG3 (20 μM) andMYBMIM (20 μM) for 6 hours. Microscopy performed here with 40×magnification and using immersion oil (scale bar indicates 20 microns).(d) Heatmap showing MYBMIM-induced gene expression changes in MOLM13cells after 6 hour treatment with TG3 (20 μM) and MYBMIM (20 μM).Heatmap is generated with values and corresponding color index (shownhere in gray scale) that are row-normalized. Biological triplicatesamples are indicated as 1-3 per sample treatment condition. (e) Volcanoplot of MYBMIM treated MV411 human AML cells compared to control. Thex-axis is denoted as the Fold change of gene expression between MYBMIMand control (log 2) and the y-axis is the Adjusted p-value (log 10).Representative MYB-dependent genes, MYC and BCL2, are significantlydownregulated. (f) BCL2 and MYC mRNA expression was measured by RT-qPCRand normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)expression. Symbols indicate p<0.05 (*p=0.0046, **p=0.00023, {circumflexover ( )}p=0.00082, {circumflex over ( )}{circumflex over ( )}p=0.0148).Each set of bars represent, from left to right, control, TG3 and MYBMIM.(g) Ectopic expression of MSCV-Ires-GFP vector containing BCL2 in MV411cells partially rescues MYBMIM-induced apoptosis, shown here bymeasurement of ATP activity using a CellTiter Glo luminescence assay. %Cell viability is calculated as a fraction of untreated control. Symbolsindicate (*p=0.00026, **p=0.00005). Error bars represent standarddeviation of the mean of 3 biological replicates. Each set of barsrepresent, from left to right, control, TG3 and MYBMIM.

Example 3. Peptide Exhibits Anti-Leukemic Efficacy In Vivo

The anti-leukemic therapeutic efficacy of the MYBMIM peptide wasinvestigated using a primary patient derived xenograft model. In thismodel, 5×10⁵ cells of a primary MLL-rearranged AML were transplanted viatail vein injection into sublethally irradiated NSG mice. In onexenograft model, after peripheral blood engraftment was confirmed (rangeof 1-7% hCD45), mice were randomized to cohorts (n=15) of eithertreatment with MYBMIM (25 mg/kg daily) or vehicle, all viaintraperitoneal injection for 21 days. At the end of the treatmentperiod, bilateral femurs were harvested, formalin fixed, paraffinembedded, and sectioned for Immunohistochemical staining of hCD45. Inthis analysis of bone marrows using hCD45 staining, MYBMIM induces adecrease in the leukemia burden in mice. Representative images are shownusing 40× magnification (FIG. 4b ). In a subsequent xenograftexperiment, 5×105 cells of a primary MLL-rearranged AML weretransplanted via tail vein injection into sublethally irradiated NSGmice. After a 3-day post-transplant period, mice were randomized tocohorts (n=15) of either treatment with MYBMIM (50 mg/kg daily) orvehicle, all via intraperitoneal injection. The endpoint analysis ofthis in vivo model was a survival analysis where MYBMIM prolongssurvival of mice, Kaplan-Meier survival curve log-rank analysis ofp=0.0038 (FIG. 4c ).

Detailed description of FIG. 3: MYBMIM exhibits anti-leukemia efficacyin vivo. (a) MYBMIM has no significant effect on colony formation ofCD34-enriched hematopoietic progenitor cells isolated from humanumbilical cord blood and grown in growth-factor enriched semi-solidmedia. The number of burst forming units-erythroid (BFU-E) and colonyforming units-Granulocyte/Monocyte (CFU-GM) and were measured here incontrol, TG3 and MYBMIM treatment conditions. The bottom section of eachbar represents CFU-GM, and the top section BFU-E. A study of MYBMIMtoxicity in NSG mice was performed with 25 mg/kg daily intraperitonealdosing for 7 days. Complete blood count (CBC) analysis was obtained fromperipheral blood samples by using a HemaVet950 with no significantdifferences in MYBMIM treated mice versus vehicle control in both (b)white blood cell counts, WBC, measured in thousands/μL and in (c)hemoglobin, measured in g/dL, n=5 for each treatment group. (d) Survivalanalysis of primary patient-derived MLL-rearranged leukemia cells(5×10{circumflex over ( )}5 cells per mouse via tail vein injection)engrafted into sublethally irradiated immunodeficient mice and treatedwith MYBMIM via intraperitoneal injection for 14 days (indicated asTreatment from days 3-17), p=0.0038. Error bars represent standarddeviation of the mean of 3 biological replicates.

Example 4. Additional Data on Peptides

Using the methods as described above, the activity of the peptides ofSEQ ID NOs:1 and 25 were evaluated in a cell viability assay using AMLcell line MV411. Quantification of live AML cells using Trypan blueexclusion after MYBMIM treatment. Live MV411 cells were counted on days2, 4, and 6 using a hemocytometer. Peptide treatment was applieddirectly to cell culture with SEQ ID NO:1 (MYBMIM; 4a) and SEQ ID NO:25(CRYBMIM; 4b) (dosed at 10 and 20 μM) every 48 hours for a 6 day period.Cells were plated and treated in biological triplicates. These peptideswere tested at 20 μM dose across a panel of AML cell lines and theresults from day 6 of treatment are shown in FIG. 4c for SEQ ID NO:1 andin FIG. 4d for SEQ ID NO:25. Each cell line control was set at 1 (blackline), and cell viability for each cell line is plotted as a fraction ofits respective control.

1. The single chain peptide of claim 3 comprising the D-amino acidsequence KLENETSMLLLELKRYpSPRRSLIERRGGRRRQRRKKRGY (SEQ ID NO:25).
 2. Thesingle chain peptide of claim 3 wherein the N-terminus has an acetylgroup, the C-terminus has an amide group, or the combination thereof. 3.A single chain peptide consisting of D-amino acids and comprising aN-terminal portion sequence selected from KLENETSMLLLELEKIRK (SEQ IDNO:19), KLENETSMLLLEL (SEQ ID NO:24) and KLENETSML (SEQ ID NO:35), aC-terminal portion sequence is RRRQRRKKRGY (SEQ ID NO:17), and thesequence therebetween selected from PADSSLDNLIKRYpSPRRSLIERR (SEQ IDNO:33), KRYpSPRRSLIERR (SEQ ID NO:23), NLIKRYpS (SEQ ID NO:36) andPADSSLDNLIKRYpS (SEQ ID NO:34).
 4. The single chain peptide of claim 3wherein a linker is present between a sequence.
 5. The single chainpeptide of claim 4 wherein the linker is GG.
 6. The single chain peptideof claim 3 wherein the N-terminus has an acetyl group, the C-terminushas an amide group, or the combination thereof.
 7. A single chainpeptide consisting of L-amino acids and comprising a N-terminal portionsequence YGRKKRRQRRR (SEQ ID NO:16), a C-terminal portion selected fromKRIKELELLLMSTENELK (SEQ ID NO:18), LELLMSTENELK (SEQ ID NO:21) andLMSTENELK (SEQ ID NO:31), and the sequence therebetween selected fromRREILSRRPpSYRKILNDLSSDAP (SEQ ID NO:29), RREILSRRPpSYRK (SEQ ID NO:20),pSYRKILN (SEQ ID NO:37) and SYRKILNDLSSDAP (SEQ ID NO:30).
 8. The singlechain peptide of claim 7 wherein a linker is present between a sequence.9. The single chain peptide of claim 8 wherein the linker is GG.
 10. Thesingle chain peptide of claim 7 wherein the N-terminus has an acetylgroup, the C-terminus has an amide group, or the combination thereof.11. The single chain peptide of claim 7 comprisingYGRKKRRQRRRGGRREILSRRPpSYRKLELLLMSTENELK (SEQ ID NO:22). 12.-42.(canceled)
 43. A composition comprising a single chain peptide of claim3 and a carrier, excipient or diluent.
 44. A method for treating canceror a dysproliferative disease comprising administering to a patient inneed thereof an effective amount of a single chain peptide of claim 3.45. The method of claim 44 wherein the cancer is acute lymphoblasticleukemia or acute lymphocytic leukemia, acute myeloid leukemia, orchronic myeloid leukemia (CML).
 46. The method of claim 44 wherein thecancer is lymphoma, small cell lung cancer, renal cell carcinoma,adenoid cystic carcinoma, squamous cell carcinoma of the head and neck,neuroblastoma, pancreatic cancer, follicular lymphoma, mantel celllymphoma, breast cancer, uterine cancer, ovarian cancer, hepatocellularcarcinoma, lung cancer, germ cell tumor, non-small cell lung cancer,gastric cancer, renal cancer, Kaposi's sarcoma, mesothelioma,desmoplastic small round cell tumor, Ewing sarcoma or lungadenocarcinoma.
 47. A composition comprising a single chain peptide ofclaim 7 and a carrier, excipient or diluent.
 48. A method for treatingcancer or a dysproliferative disease comprising administering to apatient in need thereof an effective amount of a single chain peptide ofclaim
 7. 49. The method of claim 48 wherein the cancer is acutelymphoblastic leukemia or acute lymphocytic leukemia, acute myeloidleukemia, or chronic myeloid leukemia (CML).
 50. The method of claim 48wherein the cancer is lymphoma, small cell lung cancer, renal cellcarcinoma, adenoid cystic carcinoma, squamous cell carcinoma of the headand neck, neuroblastoma, pancreatic cancer, follicular lymphoma, mantelcell lymphoma, breast cancer, uterine cancer, ovarian cancer,hepatocellular carcinoma, lung cancer, germ cell tumor, non-small celllung cancer, gastric cancer, renal cancer, Kaposi's sarcoma,mesothelioma, desmoplastic small round cell tumor, Ewing sarcoma or lungadenocarcinoma.